Mutated herpes simplex virus type 1 thymidine kinases and uses thereof

ABSTRACT

The present invention provides new site-specific HSV-thymidine kinase mutants with improved nucleoside analog metabolizing activity due to low or no thymidine phosphorylation ability. Also provided is a method of killing target cells using such mutants combined with a prodrug.

CROSS-REFERENCE TO RELATED APPLICATION

This is a divisional application of the U.S. Ser. No. 09/338,308, filedon Jun. 22, 1999 now U.S. Pat. No. 6,245,543.

This patent application claims benefit of provisional patent applicationU.S. Serial No. 60/090,271, filed Jun. 22, 1998, now abandoned.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates generally to the field of molecularbiology of herpes simplex viruses and vaccine technology. Morespecifically, the present invention relates to a means of improving genetherapy for diseases such as cancer by mutating herpes simplex virustype 1 thymidine kinases and uses thereof.

2. Description of the Related Art

The herpes simplex virus thymidine kinases (HSV-TKs)¹ are thepharmacological targets of most herpesvirus treatments (1, 2), and morerecently, HSV-1 TK has been utilized as a suicide gene therapeutic forcancer in combination with ganciclovir (3, 4). The basis for these usesis their ability to specifically phosphorylate anti-herpesvirusnucleoside drugs such as acyclovir (ACV), ganciclovir (GCV) and5-bromovinyldeoxyuridine (BVDU) (1, 2, 5). This targeting is basedprimarily on the differences in substrate specificity compared to thecellular TKs. The HSV-1 TK has a much broader range of substrates whichinclude most pyrimidine nucleosides, many guanosine derivatives (e.g.,ACV or GCV), and most purine and pyrimidine nucleoside triphosphates(6-9). HSV-TK also possesses a thymidylate kinase (TMPK) activity, butthis activity is restricted to only deoxypyrimidine monophosphatesubstrates (7-9).

Proteolytic mapping studies of HSV-1 TK with the photoactive TMP analog,[³²P]5N₃dUMP, identified a region of the thymine base binding siteinclusive in the peptide Ile¹¹²-Tyr¹³² (10). This report, and others (7,8, 11, 12), concluded that the thymine base of TMP and thymidine bind inone shared site. This was subsequently confirmed in comparisons of twoX-ray crystal structures of HSV-1 TK with bound thymidine or TMP (13,14). Two initial X-ray crystal structures of HSV-1 TK have beenpublished (13, 14), one with bound thymidine or ganciclovir (13) and theother with thymidine, 5-iodo-deoxyuridine monophosphate or a complexwith TMP and ADP (14). Subsequent structures have been reported withbound acyclovir, penciclovir and other nucleoside drug substrates andinhibitors (15,16). Within the pyrimidine base binding site, allstructures have indicated that hydrogen bonding between Gln-125 of HSV-1TK and the N3 and O4 atoms of the pyrimidine base was evident (13-15).In the complex with ganciclovir or acyclovir, Gln-125 was shown to formhydrogen bonds with the N1 and O6 atoms of the guanine base of GCV (13,15, 16).

The prior art is deficient in lack of improved mutants of herpes simplexvirus type 1 thymidine kinases useful in treating cancers in genetherapy techniques so as to maximize therapeutic efficacy and minimizeuntoward side effects. Increasing and/or modifying the desired substratespecificity for HSV-thymidine kinase would ameliorate these sideeffects. The present invention fulfills this long-standing need anddesire in the art.

SUMMARY OF THE INVENTION

It has been reported that site-directed mutagenesis of Gln-125 to Glu,Leu or Asn can modulate the substrate affinities for thymidine and ACVin the context of HSV-1 TK in antiviral drug resistance (17). To examinethe role of Gln-125 in HSV-1 TK activity in the context of genetherapeutic applications, three separate site-specific mutations weremade of this residue to either an Asp, Asn or Glu acid residue. Thesethree mutants and wild-type HSV-1 TK were expressed in E. coli,partially purified, and then were compared for their ability tophosphorylate deoxypyrimidine and acyclic purine substrates. For eachmutation, the ability to phosphorylate deoxypyrimidine substrates weregreatly modified, while activity for the acyclic purines was variable.Kinetic constants for thymidine and GCV were also determined. Themolecular basis for the obtained results were evaluated using Flexidockmolecular modeling simulations of the different enzyme active sites. Thegenes for each mutant HSV-1 TK were incorporated into a retroviralplasmid for expression in two mammalian cell lines and evaluation ofsensitivity to GCV killing. The potential uses of these mutants in genetherapy applications and in the design of new HSV-1 TK proteins withdifferent activities is discussed.

In one embodiment of the present invention, there is provided a mutantherpes simplex virus type 1 thymidine kinase protein with asite-specific mutation at amino acid position 125 of wild type herpessimplex virus type 1 thymidine kinase.

In another embodiment of the present invention, there is provided avector comprising a DNA sequence coding for the mutant herpes simplexvirus type 1 thymidine kinase protein disclosed herein, a promoter andoptionally an origin of replication.

In still another embodiment of the present invention, there is provideda host cell transfected with the above disclosed vector.

In still yet another embodiment of the present invention, there isprovided a method of killing target cells, comprising the steps oftransfecting or transducing the target cells with a gene encoding anon-human mutant herpes simplex virus type 1 thymidine kinase and thencontacting the transfected or transduced cells with an effective amountof a prodrug, wherein the prodrug is a substrate for the mutant herpessimplex virus type 1 thymidine kinase to yield a toxic substance, whichinhibits cellular DNA polymerases and kills the transfected ortransduced target cells.

Other and further aspects, features, and advantages of the presentinvention will be apparent from the following description of thepresently preferred embodiments of the invention. These embodiments aregiven for the purpose of disclosure.

BRIEF DESCRIPTION OF THE DRAWINGS

So that the matter in which the above-recited features, advantages andobjects of the invention, as well as others which will become clear, areattained and can be understood in detail, more particular descriptionsof the invention briefly summarized above may be had by reference tocertain embodiments thereof which are illustrated in the appendeddrawings. These drawings form a part of the specification. It is to benoted, however, that the appended drawings illustrate preferredembodiments of the invention and therefore are not to be consideredlimiting in their scope.

FIG. 1 shows autoradiograph of photolabeled wild-type and mutant HSV-1TKs. Partially purified extracts from each HSV-1 TK were photolabeledwith the TMP photoaffinity analog, [³²P]5-azido-dUMP. Photolabeledproteins were separated on 10% SDS-polyacrylamide gels followed byautoradiography of the dried gel.

FIG. 2 shows Flexidock modeling of hydrogen bonding between thymine andamino acid 125. The Flexidock module of SYBL 6.3 was used to generatethe lowest energy conformations of bound thymidine with wild-typeGln-125 HSV-TK (FIG. 2A), or the mutant HSV-1 TKs, Glu-125 (FIG. 2B),Asp-125 (FIG. 2C) or Asn-125 (FIG. 2D).

FIG. 3 shows sensitivity to GCV killing of wild-type and mutant HSV-1 TKexpressing HCT-116 and NIH3T3 cell lines. HCT-116 and NIH3T3 cell linesstably expressed wild-type or mutant HSV-1 TKs under control of aMoloney murine leukemia virus promoter. Cells were plated in triplicateand exposed to 0, 0.05, 0.5 or 5 μM GCV for four days. Cell viabilitieswere determined using MTT dye. Results are presented as percent ofuntreated cell controls for each cell line. FIG. 3A: NIH3T3 cell lines,FIG. 3B: HCT-116 cell lines; parental cells (noTK, dark bands); Asp125TK(Asp-#, brick band); Glu-125TK (Glu-#, cross-hatch bands); Asn-125TK(Asn-#, gray bands); wild-type HSV-1 TK (WT-#, striped bands).

FIG. 4 shows clonal dilution assays for GCV sensitivity. ParentalHCT-116 (large square), wild-type HSV-1 TK cells (triangle), Asn-125TKcells (small square) and Glu-125TK cells (diamond) were treated with 0,0.1, 1 or 10 μM GCV in triplicate for 24 hours. After this time, eachwell of cells was sequentially diluted from 1:10 to 1:10,000 in 1 ml offresh media on a separate 24 well plate. After 7 days, surviving cellcolonies were fixed in 100% methanol, stained with 0.1% methylene blueand counted.

FIG. 5 shows clonal dilution assays for bystander effect cell killing.Each of the three HSV-1 TK expressing cell lines were plated withparental HCT-116 cells (total 2×10⁵/well) in the following proportions:(parental: HSV-1TK cells) 100%:0; 95%:5%; 90%:10%; 75%:25%; 50%:50% and0:100%. Cells were treated with 25 mM GCV for 24 hours, then each wellof cells was sequentially diluted from 1:10 to 1:10,000 in 1 ml of freshmedia on a separate 24 well plate. After 7 days, surviving cell colonieswere fixed in 100% methanol, stained with 0.1% methylene blue andcounted.

FIG. 6 shows cell cycle analysis of GCV treated cells. Parental HCT-116cells and each HSV-1 TK expressing cell line were grown to 60%confluency in 25 cm² flasks and treated for 24 hours minus (−GCV, panelsA, C, E and G) or plus (+GCV, panels B, D, F and H) 25 μM GCV. Cellswere fixed in 70% ethanol overnight, and later incubated with RNAase(0.1%) and propidium iodide (1 mg/ml) for 20 minutes on ice. Flowcytometry measuring propidium iodide fluorescence was done with aBecton-Dickinson FACScalibur instrument using MODFIT computer software.FIGS. 6A and 6B: HCT-116 cells; FIGS. 6C and 6D: wild-type HSV-1 TKcells; FIGS. 6E and 6F: Asn-125TK cells, FIGS. 6G and 6H: Glu-125TKcells.

FIG. 7 shows GCV-induced apoptosis in HSV-TK-expressing cell lines. TheHCT-116 cells expressing wild-type HSV-1 TK (FIG. 7A), Asn-125TK (FIG.7B), or Glu-125TK (FIG. 7C) were grown on chamber slides and incubatedwith 25 μM GCV for 36 or 84 hours. Control cultures for each cell linewithout GCV addition (top row) were evaluated after 84 hours. At theindicated time point, cells were fixed in methanol, stained with DAPI,and nuclei visualized by fluorescent microscopy. Each image is magnified40×.

FIG. 8 shows activation of Caspase 3 in response to GCV treatment.Parental HCT-116, wild-type HSV-1TK, Asn-125TK and Glu-125TK-expressingHCT-116 cells were treated with 0 (black bars) or 25 μM GCV (gray bars)for 4 days. Cell lysates were prepared and caspase 3 assays performedusing a colorimetric ApoAlert CPP32/Caspase-3 kit as per manufacturersinstructions (Clontech). Relative caspase 3 activity was determined byO.D. 405 nm readings of the cleaved DEVD-pNA substrate and presented asDVEDase activity.

DETAILED DESCRIPTION OF THE INVENTION

The herpes simplex virus type-1 thymidine kinase (HSV-1 TK) is the majoranti-herpesvirus pharmacological target, and it is being utilized incombination with the prodrug, ganciclovir, as a suicide gene therapeuticfor cancer. One active-site amino acid, glutamine-125 (Gln-125), hasbeen shown to form hydrogen bonds with bound thymidine, thymidylate andganciclovir in multiple X-ray crystal structures. To examine the role ofGln-125 in HSV-1 TK activity, three site-specific mutations of thisresidue to either an aspartic acid, asparagine or glutamic acid wereintroduced. These three mutants and wild-type HSV-1 TK were expressed inE. coli, partially purified, and their enzymatic properties compared. Incomparison to the Gln-125 HSV-1 TK, TMPK activity was abolished in allthree mutants. For thymidine kinase activity relative to Gln-125 enzyme,the K_(m) of thymidine increased from 0.9 μM for the parent Gln-125enzyme to 3 μM for the Glu-125 mutant, to 6000 μM for the Asp-125 mutantand to 20 μM in the Asn-125 mutant. In contrast, the K_(m) ofganciclovir decreased from 69 μM for the parent Gln-125 enzyme to 50 μMfor the Asn-125 mutant, and increased to 473 μM for the Glu-125 mutant.The Asp-125 enzyme was able to poorly phosphorylate ganciclovir, butwith non-linear kinetics. Molecular simulations of the wild-type andmutant HSV-1 TK active-sites predict that the observed activities aredue to loss of hydrogen bonding between thymidine and the mutant aminoacids, while the potential for hydrogen bonding remains intact forganciclovir binding. When expressed in two mammalian cell lines, theGlu-125 mutant led to GCV mediated killing of one cell line, while theAsn-125 mutant was equally effective as wild-type HSV-1 TK inmetabolizing GCV and causing cell death in both cell lines.

The method of treatment basically consists of providing to target cellsthe mutant herpes simplex virus type 1 thymidine kinase gene and thenexposing the cells to an appropriate substrate which is converted to atoxic substance to kill those cells expressing the mutant HSV-1thymidine kinase gene as well as those in the vicinity of the mutantHSV-1 thymidine kinase gene-expressing cells, i.e., bystander cells. Themutant HSV-1 thymidine kinase gene can be administered directly to thetargeted cells or systemically in combination with a targeting means,such as through the selection of a particular viral vector or deliveryformulation. Cells can be treated in vivo, within the patient to betreated, or treated in vitro, then injected into the patient. Followingintroduction of the mutant HSV-1 thymidine kinase gene into cells in thepatient, the prodrug is administered, systemically or locally, in aneffective amount to be converted by the mutant HSV-1 thymidine kinaseinto a sufficient amount of toxic substance to kill the targeted cells.A nucleoside analog which is a substrate for HSV-1 TK to produce a toxicsubstance which kills target cells is referred to herein as a “prodrug”.

The destruction of selected populations of cells can be achieved bytargeting the delivery of the mutant HSV-1 thymidine kinase gene. Thenatural tropism or physiology of viral vectors can be exploited as ameans of targeting specific cell types. For example, retroviruses arewell known to become fully active only in replicating cells. This facthas been used as the basis for selective retroviral-mediated genetransfer to replicating cancer cells growing within a site where thenormal (nonmalignant) cells are not replicating, in both animal andhuman clinical studies. Alternatively, the viral vector can be directlyadministered to a specific site such as a solid tumor, where the vastmajority of the gene transfer will occur relative to the surroundingtissues. This concept of selective delivery has been demonstrated in thedelivery of genes to tumors in mice by adenovirus vectors. Molecularconjugates can be developed so that the receptor binding ligand willbind only to selective cell types, as has been demonstrated for thelectin-mediated targeting of lung cancer.

Recently, it was shown that intravenous injection of liposomes carryingDNA can mediate targeted expression of genes in certain cell types.Targeting of a gene encoding a mutant HSV-1 thymidine kinase orexpression of the gene to a small fraction of the cells in a tumor massfollowed by substrate administration could be adequate to mediateinvolution.

Another example of protein delivery to specific targets is that achievedwith liposomes. Methods for producing liposomes are described (e.g.,Liposomes: A Practical Approach). Liposomes can be targeted to specificsites by the inclusion of specific ligands or antibodies in theirexterior surface, e.g. targeting of specific liver cell populations byinclusion of asialofetuin in the liposomal surface (43). Specificliposomal formulations can also achieve targeted delivery, as bestexemplified by the so-called Stealth' liposomes that preferentiallydeliver drugs to implanted tumors (44). After the liposomes have beeninjected or implanted, unbound liposome is allowed to be cleared fromthe blood, and the patient is treated with the prodrug. Again, thisprocedure requires only the availability of an appropriate targetingvehicle.

The present invention is directed to a novel means of improving upon thesafety and efficacy of gene therapy, particularly for neoplasticdisease. The invention provides mutant herpes simplex virus type 1thymidine kinase proteins for use in gene therapy for cancer and viraldiseases.

In one embodiment of the present invention, there is provided a mutantherpes simplex virus type 1 thymidine kinase protein, which contains asite-specific mutation at the amino acid position 125, i.e., glutamineresidue of the wild type herpes simplex virus type 1 thymidine kinaseprotein. Preferably, the glutamine residue is mutated to a non-glutamineresidue selected from the group consisting of glutamic acid, asparticacid and asparagine.

The mutant herpes simplex virus type 1 thymidine kinase proteins of thepresent invention provide for improved metabolizing activity ofnucleoside analogs such as ganciclovir, due to low or no endogenousthymidine phosphorylation ability, leading to enhanced therapeuticefficacy of the analogs.

The present invention is also directed to a vector comprising a DNAsequence coding for the mutant herpes simplex virus type 1 thymidinekinase protein disclosed herein and the vector is capable of replicationin a host which comprises, in operable linkage: a) optionally, an originof replication; b) a promoter; and c) a DNA sequence coding for themutant protein. Preferably, the vector is selected from the groupconsisting of a retroviral vector, an adenoviral vector, anadeno-associated viral vector, a herpes vector, a viral vector and aplasmid.

The present invention is also directed to a host cell transfected withthe vector of the present invention so that the vector expresses amutant herpes simplex virus type 1 thymidine kinase protein. Preferably,such host cells are selected from the group consisting of bacterialcells, mammalian cells and insect cells.

In another embodiment of the present invention, there is provided amethod of killing target cells in an individual in need of suchtreatment, comprising the steps of transfecting or transducing thetarget cells with a gene encoding a mutant herpes simplex virus type 1thymidine kinase and then contacting the transfected or transduced cellswith an effective amount of a prodrug, wherein the prodrug is asubstrate for the mutant herpes simplex virus type 1 thymidine kinaseand may be administered systemically or locally. The substrate isnon-toxic to the target cells and is phosphorylated by the kinase toyield a toxic substance which inhibits cellular DNA polymerases andkills the transfected or transduced target cells. Representativeexamples of the prodrugs are nucleoside analogs acyclovir, ganciclovirand 5-bromovinyldeoxyuridine. Preferably, the targeted cells areselected from the group consisting of tumor cells and virally infectedcells.

The following examples are given for the purpose of illustrating variousembodiments of the invention and are not meant to limit the presentinvention in any fashion.

EXAMPLE 1

Materials

All reagents and nucleotides were purchased from Sigma Chemical Co.unless otherwise indicated. [³H-methyl]thymidine (71 Ci/mmol),[³H-methyl]AZT (19 Ci/mmol), [8-³H]ganciclovir (17 Ci/mmol) and[8-³H]acyclovir (15 Ci/mmol) were purchased from Moravek Biochemicals.[5-³H]deoxycytidine (20 Ci/mmol) was from American RadiolabeledChemicals. [γ-³²P]ATP was from ICN Radiochemicals. Whatman DE81 filterpaper discs and DE-52 resin were purchased from Fisher. Automated DNAsequencing of plasmids was done by the U.A.M.S. Molecular ResourceLaboratory using a Model 377 DNA Sequencer from Applied Biosystems.Oligonucleotide primers were synthesized in the U.A.M.S. MolecularBiology Core Facility. Restriction endonucleases, Vent polymerase, andT4 DNA ligase were from New England Biolabs.

EXAMPLE 2

Mutagenesis and Expression Vector Constructions

Starting with a previously prepared pET-9a (Novagen) HSV-1 thymidinekinase construct pET-TK1 (12), a 1.5 kb SalI-BamHI fragment was excisedand subcloned into a pUC118 vector (pUC118-TK). Single-strandedpUC118-TK DNA was purified from cultures of JM107 after infection withhelper phage M13K07 as previously described (18). Three mutagenicoligonucleotide primers were prepared to replace Gln-125 with eitherAsp-125, Asn-125 or Glu-125 as follows: ACAAGCGCCGACATAACAATG (SEQ IDNo. 1) for Asp, ACAAGCGCCAACATAACAATG (SEQ ID No. 2) for Asn andACAAGCGCCGAAATAACAATG (SEQ ID No. 3) for Glu. The three primers wereused with an Amersham Sculptor in vitro mutagenesis kit as permanufacturers directions as adapted from Sayers, et al. (19). Plasmidsisolated from the resulting colonies were sequenced by automatedsequencing to confirm the presence of the mutation and its identity.After confirmation of each mutation, the DNA pieces were released bySalI-BamHI restriction digests and re-ligated back into the originalpET-TK1 expression plasmid for transformation. The resulting colonieswere again sequenced to confirm the presence of the mutated residues.

EXAMPLE 3

Enzyme Purification

E. coli BL21SY211 cells, in which T7 RNA polymerase is under control ofthe IPTG inducible lacUV5 promoter (20), were transformed with eachpET-TK plasmid. These cells were grown to A₆₀₀=0.6, induced by 1 mM IPTGfor 2.5 hours, and lysed in buffer A (20 mM Tris buffer, pH 8.1, 10%glycerol, 1 mM DTT, 40 mM KCl, 1 mM EDTA, 1 mM PMSF, and aprotinin (1μg/ml)) by sonication for 3 minutes. The homogenate was then centrifugedat 10,000×g for 30 minutes to separate insoluble material. The solublelysate was loaded onto a column of DE-52 in tandem sequence with aheparin agarose column (both 15×2 cm) and washed extensively with bufferA. The heparin agarose column was uncoupled from the DE-52 column andbound proteins were eluted in 50 ml buffer A plus 300 mM NaCl. Theresulting fraction with thymidine kinase activity was concentrated toapproximately 10 ml in an Amicon filtration apparatus, diluted withthree volumes of buffer A (minus NaCl), and re-concentrated. Thisapproach has reproducibly led to greater than 80% purified HSV-TK asdetermined by Coomassie blue staining with cumulative yields rangingfrom 0.5 to 2 mg total protein. The stability of the mutant enzymepreparations relative to wild-type preparations were identical whenstored at −20° C. for up to 6 weeks (data not shown).

EXAMPLE 4

Enzyme Assays

The activity of the purified HSV-1 TK was determined using the followingstandard reaction mixture for thymidine as a substrate (12): 3 μg ofprotein, 4 μM [³H-methyl]thymidine (0.1 mCi/mmol), 20 mM potassiumphosphate, pH 7.6, 1 mM DTT, 5 mM ATP, 5 mM MgCl₂, 25 mM NaF, 40 mM KCl,and 0.5 mg/ml BSA in a total volume of 25 μl for 10 minutes at 37° C. Toidentify phosphorylated products, 20 μl of the reaction mixture wasloaded onto a DE-81 filter and dried, washed once in 1 mM ammoniumformate, twice more in 95% ethanol, and counted for radioactivity (12).Radioactivity was determined using an LKB 1214 Rackbeta liquidscintillation counter and corrected for background using controls withenzyme incubated in the absence of ATP. For TMPK activity, 20 μM[³H-methyl]TMP was substituted for thymidine in the above assay mixture,and [³H]TDP product was determined by separation from [³H]TMP on thinlayer PEI-cellulose plates developed in 0.35M LiCl (21). Amount ofproduct converted to TDP was quantitated by scintillation counting ofthe excised TMP and TDP spots. The filter binding assay was used toquantitate the phosphorylation of [³H]dC (200) μM and [³H]AZT (200 μM)with the four enzymes. To compare relative enzymatic activities for[³H]GCV (50 μM) and [³H]ACV (50 μM), reactions were loaded onto smallDE-52 columns packed in Pasteur pipettes as previously described (22).Unreacted nucleoside substrates do not bind to this resin, andphosphorylated products were eluted in 100 mM ammonium bicarbonate andquantified for radioactivity.

For determination of K_(m) values of thymidine and GCV for the fourenzymes, the filter binding assay was utilized. The basic reactioncomponents were as above, and the following enzyme concentrations,substrate ranges and times were used under linear initial velocityconditions: for [³H]thymidine: parent Gln-125TK: 0.1 μg TK, 0.1-4 μM, 10min; Asn-125TK: 0.2 μg TK, 0.25-10 μM, 15 min; Asp-125TK: 4 μg TK,0.1-2.5 μM, 30 min; Glu-125TK 0.5 μg TK, 0.5-2.5 μM, 15 min. For[³H]GCV: parent Gln-125TK: 2 μg TK, 5-100 μM, 15 min; Asn-125TK: 0.6 μgTK, 0.5-15 μM, 15 min; Asp-125TK: Not determined; Glu-125TK 4 μg TK,50-1000 μM, 25 min. Each reaction was done in triplicate. Forcalculation of kinetic constants, non-linear regression Michaelis-Mentenanalyses were done using PSI-PLOT version 5.0 (Poly SoftwareInternational).

EXAMPLE 5

Photoaffinity Labeling of HSV-1 TK

The TMP photoaffinity analog, [³²P]5-N₃dUMP, was synthesizedenzymatically using HSV-1 TK, 5-azido-deoxyuridine and [g-³²P]ATP aspreviously described (10, 12). For photolabeling studies, 10 μg of theDE-52/heparin agarose purified HSV-TKs were incubated with 10 mM[³²P]5-N₃dUMP for 10 seconds. The sample was then irradiated for 90seconds with a hand-held UV lamp (254 nm UVP-11, Ultraviolet Products,Inc.) at a distance of 3 cm. Reactions were terminated by addition of anequal volume of 10% TCA, incubated on ice for 10 minutes, and pelletedby centrifugation at 13,000×g for 5 minutes. The protein was resuspendedin a solubilization mixture (23) and separated on 10% SDS-polyacrylamidegels. Dried gels were exposed to film for 2 days.

EXAMPLE 6

Molecular Modeling of Substrate Interactions with HSV-1 TK

The active-site region of wild-type HSV-1 TK with bound thymidine or GCVwas excised from the coordinates for the X-ray crystal structure ofHSV-1 TK (kindly provided by Dr. Mark Sanderson (14)) and loaded intothe Flexidock module of the molecular modeling program, SYBIL 6.3.Because the molecular interactions between bound thymidine and GCV weresimilar in the crystal structures except for rotation of Gln-125, thisresidue was defined as movable in the Flexidock program. As a controlfor this assumption, thymidine was placed in the Gln-125 HSV-1 TK activesite and the lowest energy conformation determined. The lowest energyconformation was identical to that reported for thymidine in the crystalstructure (14). Thus, lowest energy conformations for bound thymidineand GCV were done for the Gln-125, Glu-125, Asp-125 and Asn-125 activesites.

EXAMPLE 7

Expression and Characterization of HSV-1 TKs in Cell Lines

A Moloney murine leukemia virus derived plasmid for the expression ofHSV-TK, termed pLENTK, has been previously constructed (24). A uniqueBspEI-MluI restriction fragment within the HSV-TK sequence of pLENTKcontains the Gln-125 mutation site. This fragment was removed fromwild-type plasmid and replaced with the analogous fragments encodingeach mutant. The new pLEN-mutant-TK constructs were sequenced to confirmthe presence of the mutation. Along with wild-type HSV-1 TK plasmid,each mutant-TK plasmid was transfected individually into the murinefibroblast cell line, NIH 3T3, and the human colon tumor cell line,HCT-116, using lipofectin reagent (GIBCO/BRL) (2 μg plasmid, 14 μl lipidper 1×10⁶ cells). Cells were maintained in RPMI 1640 media and selectedwith G418 (200 μg/ml for 2 weeks) as previously described (24). At leasteight individual G418 resistant cell clones were picked and grown up forfurther characterization. Each clone was screened initially for growthinhibition by 25 μM GCV. Those clones that were sensitive were furtheranalyzed for HSV-TK protein expression by Western blot analysis with apolyclonal, rabbit anti-HSV-TK antibody (a gift from Dr. MargaretBlack). For each clone, cell numbers were normalized to 1×10⁶, and equalprotein loading was confirmed for each sample by gel staining. BlottedHSV-1 TK protein bands were visualized on film using ECL chromophorereagents (Amersham). For analysis of GCV sensitivity of differentclones, NIH3T3 and HCT-116 cell sets were seeded in 96 well plates in0.1 ml media (15,000 cells/well). The next day, a dose range of GCV(0.005 to 5 mM, n=3) was added in 0.1 ml media. After 4 days, MTT (50mg/well) was added for 1.5 hours, followed by DMSO solubilization of thecells and absorbance reading at 540 nm (25).

EXAMPLE 8

Metabolic Labeling with [³H]Nucleosides

For metabolic labeling, cells (1-2×10⁶) were labeled in triplicate with2 μCi [³H]GCV (8 mM) for 18 hours, then nucleotides were extracted frompelleted cells in 0.2 ml 70% methanol at 4° C. for 15 minutes aspreviously described (21, 24). An aliquot of each methanol solublesupernatant was analyzed for radioactivity by scintillation counting.The methanol insoluble pellets, representative of a crude DNA fraction,were resuspended in 0.15 ml water and also counted for radioactivity.For deoxpyrimidines, cells were grown to confluency in 60 cm² plates,and either 2 μCi [³H]thymidine (2 μM final) or 2 μCi [³H]dC (10 μMfinal) were added for 1 or 2 hours respectively prior to extraction in70% methanol. Methanol-soluble extracts were concentrated by evaporationunder nitrogen, and separated on PEI-cellulose thin layer chromatographyplates developed in 0.8 M LiCl for GCV or 0.35 M LiCl for thymidine/dCas previously described (21).

EXAMPLE 9

GCV Sensitivity and Bystander Effect Clonal Dilution Assays

For determination of GCV sensitivity, parental HCT-116 and each HSV-1 TKexpressing cells were seeded in 24 well plates (2×10⁵/well) in 1 ml ofmedia. The next day, 0, 0.1, 1 or 10 μM GCV was added to each cell linein triplicate. After 24 hours, for each well the media was removed,cells were rinsed twice in fresh media, trypsinized, then media wasadded to 1 ml per well. Each well of cells was then sequentially dilutedfrom 1:10 to 1:10,000 in 1 ml of fresh media on a separate 24 wellplate. After 7 days, surviving cell colonies were fixed in 100%methanol, stained with 0.1% methylene blue and counted. For bystandereffect assays, each of the three HSV-1 TK expressing cell lines wereplated with parental HCT-116 cells (total 2×10⁵/well) in the followingproportions: (parental: HSV-1TK cells) 100%:0; 95%:5%; 90%:10%; 75%:25%;50%:50% and 0:100%. After two days, 25 μM GCV was added in 1 ml freshmedia. After 24 hrs, the media was removed and cells from each well werediluted from 1:10 to 1:10,000 as described above. After 7 days,surviving cell colonies were fixed and stained for counting.

EXAMPLE 10

DAPI-Staining of Apoptotic Cells

Parental HCT-116 cells and each HSV-1 TK expressing cell line wereplated (5×10⁴ cells/well) in 8-well plastic chamber slides (Lab-Tek) andtreated plus or minus 25 mM GCV for 36 or 84 hrs. At either time point,cells were washed with phosphate-buffered saline followed by staining in1 μg/ml DAPI (4′,6′-diamidine-2′-phenylindoledihydrochloride) in 100%methanol at 37° C. for 10 min. After rinsing, the stained cells werevisualized with a Zeiss flourescent microscope at 40× magnification witha DAPI-specific filter.

EXAMPLE 11

Caspase 3 Assay

Caspase 3-like activity was determined in parental HCT-116 and eachHSV-1 TK expressing cell line treated for 50 hrs plus or minus 25 μM GCVusing an Apo-Alert CPP32/Caspase 3 Colorimetric Assay kit with thepeptide substrate, DEVD-pNA, as per manufacturers instructions (ClontechLaboratories, INC.). GCV-treated and untreated cells were grown in 25cm² flasks, and cell numbers were determined using a hemocytometer priorto analysis. Assays were done in triplicate with protein extractsderived from 2×10⁶ cells. The amount of Caspase 3-like activities werequantitated using a Shimadzu UV/VIS spectophotometer set at 405 nm.

EXAMPLE 12

Cell Cycle Analysis

Parental HCT-116 cells and each HSV-1 TK expressing cell line were grownto 60% confluency in 25 cm² flasks and treated for 24 hours plus orminus 25 μM GCV. Following drug incubation, cells were removed bytrypsin and total cell numbers determined. Following two phosphatebuffered saline rinsing and centrifugation cycles, the cell pellets wereresuspended in 1 ml of 70% ethanol and stored at 4° C. until furtheranalysis. Just prior to cell cycle analysis, the ethanol was removed andcell pellets were resuspended in phosphate-buffered saline plus RNAase(0.1%) and propidium iodide (1 mg/ml) for 30 minutes on ice. Flowcytometry measuring propidium iodide fluorescence was done with aBecton-Dickinson FACScalibur instrument. Cell cycle distribution of thecells were determined using MODFIT computer software

EXAMPLE 13

Mutagenesis and Expression of Gln-125 Mutants of HSV-1 TK

Expression plasmids derived from wild-type pET-TK1 (12) encoding theAsp, Asn or Gln changes were individually transformed into BL21 E. coli,grown and induced with IPTG. After cell pelleting and sonication, theresulting mutant HSV-TKs, along with wild-type HSV-TK and pET9a controlpreparations, were partially purified over tandem DE-52 and heparinagarose columns. This is a modification of the previously describedpurification method for HSV-1 TK (12), in that the pH of the lysisbuffer has been changed from pH 7.6 to pH 8.1. This change allows thebulk of expressed HSV-1 TK to flow-through the DE-52 column, instead ofweakly absorbing as in the previous procedure (12). Under conditionsutilized, no TK or TMPK activities in the E. coli pET-9a extracts weredetected (data not shown).

EXAMPLE 14

Enzymatic Activities of the Gln-125 HSV-1 TK Mutants

As an initial screen for activity, each expressed HSV-1 TK enzyme wasassayed for phosphorylation of the following substrates: thymidine (4μM), TMP (20 μM), ACV (50 μM), GCV (50 μM), deoxycytidine (dC, 200 μM),or 3′-azido-2′,3′dideoxythymidine (AZT, 200 μM). Enzymatic conditionsfor optimal wild-type HSV-1 TK activities were utilized, thus resultspresented in Table 1 for each substrate were normalized to 100% valuesfor comparative purposes. As expected, the wild-type Gln-125 HSV-1 TKefficiently phosphorylated each of these substrates. For each mutant,there was a striking decrease in their ability to phosphorylatepyrimidine nucleosides, and minimal TMPK activity for the TMP substrate.For metabolism of GCV and ACV, the Asn-125TK retained most of thesephosphorylation activities, while activities for the Asp-125TK andGlu-125TK were decreased to 14% and 7% for ACV and 0.7% and 5% for GCVrespectively.

TABLE 1 Analysis of Reaction Products for Wild-type and Mutant HSV-1 TKsPRODUCT FORMATION (% of wild-type Gln-125) TK Enzyme TMP TDP GCVMP ACVMPdCMP AZTMP Gln-125 (WT) 100 100 100 100 100 100 Glu-125 43 7 5 7 0.2 48Asn-125 22 6 85 92 0.2 9 Asp-125 0.5 4 0.7 14 >0.1 8

Due to these observed differences in purine versus pyrimidinemetabolism, and because of the use of HSV-1 TK in cancer gene therapyapplications with GCV (3, 4), the K_(m) and k_(cat(app)) for thymidineand GCV were determined for each of the four HSV-1 TKs and listed inTable 2. Unlike the substrate screening assays presented in Table 1,linear velocity conditions for each enzyme and substrate wereestablished prior to K_(m) determinations. For thymidine, the K_(m)sincreased relative to wild-type enzyme approximately 3-fold for theGlu-125 enzyme, 20-fold for the Asn-125 enzyme and 6000-fold for theAsp-125 enzyme. The k_(cat) doubled for the Asp-125 and Asn-125 enzymes,while a 20-fold decrease in k_(cat) was determined for the Glu-125TK.For GCV, the K_(m) decreased from 69 μM for wild-type enzyme to 50 μMfor the Asn-125TK, while the K_(m) increased to 473 μM for the Glu-125enzyme. These mutations also caused a 6-fold and 12-fold decreaserespectively in the k_(cat) compared to wild-type enzyme. Interestingly,no linear velocity conditions could be established for the Asp-125TKwith GCV. As was shown in Table 1, this enzyme will phosphorylate asmall amount of GCV, however, it does not generate product in an initialvelocity-dependent manner. The basis for this lack of activity was notfurther evaluated.

TABLE 2 Kinetic Constants of HSV-1 TK Gln-125 Mutants for Thymidine andGanciclovir THYMIDINE GANCICLOVIR HSV-1TK K_(m) (μM) k_(cat) (s⁻¹)k_(cat) /K_(m) (M⁻¹s⁻¹) K_(m) (μM) k_(cat) (s⁻¹) k_(cat) /K_(m) (M⁻¹s⁻¹)Gln-125 0.9 0.06 6.7 × 10⁴ 69 0.47 6.8 × 10³ WT Asn-125 20 0.13 6.5 ×10³ 50 0.08 1.7 × 10³ Glu-125 3 0.003 844 473 0.04 82 Asp-125 6000 0.11 18 N.D. N.D. N.D. N.D.—not determined

EXAMPLE 15

Photoaffinity Labeling of the Gln-125 HSV-1 TK Mutants

The thymidine and TMP photoaffinity analog, [³²p]5-azido-dUMP, hasproven useful as an active-site cross-linking reagent for studying HSV-1TK (10, 12). This analog was used for photocrosslinking of the fourHSV-1 TKs. As shown in FIG. 1, [³²P]5-azido-dUMP was photoincorporatedefficiently into the wild-type Gln-125 enzyme, but only tracephotoincorporation was detected for the three mutant HSV-1 TKs. Theseresults further demonstrate the weak binding affinities of the threemutant HSV-1 TKs for pyrimidine substrates.

EXAMPLE 16

Molecular Modeling Comparisons of Thymidine and Ganciclovir in theGln-125 and Mutant HSV-1 TK Active Sites

The Flexidock component of the molecular modeling program, SYBIL 6.3,was used with the coordinates of the wild-type Gln-125 HSV-1 TK crystalstructure (14) to dock thymidine or GCV in the active site of each ofthe four HSV-1 TK enzymes. As shown in FIG. 2, loss of hydrogen bondingbetween the N3 and O4 of the thymine base and Asn-125 or Asp-125 may bethe molecular basis for the decreased pyrimidine substratephosphorylation activities of these enzymes. This analysis predicts thatthe Glu-125 enzyme may still form one hydrogen bond, thus retaining itsthymidine phosphorylation activity. Analysis of GCV in the active sitepredicts that it is still able to maintain hydrogen bonding with theAsp, Asn, or Glu residues (data not shown), and thus could contribute tothe retention of this phosphorylation activity with the Asn-125 mutant.Introduction of the negatively charged Asp and Glu residues clearlyattenuated the GCV phosphorylation activities relative to the Asnmutation, and it is thus likely that this charge difference alsocontributes to the observed changes in enzymatic properties.

EXAMPLE 17

Cellular Expression of HSV-1 TK and Sensitivity to GCV Killing

cDNA for each mutant was incorporated into a Moloney murine leukemiavirus plasmid (24). The plasmids for wild-type HSV-1 TK and each mutantwere individually transfected into either NIH3T3 cells or the humancolon tumor cell line, HCT-116. Following drug selection in G418,individual cell clones were evaluated for sensitivity to GCV andrelative levels of HSV-1 TK expression. Using an HSV-1 TK antibody andextracts normalized by protein and cell number, the relative expressionlevels of HSV-1 TK in each cell clone were determined. Cell sets havingequivalent expression of an HSV-1 TK and roughly equivalent cell growthrates were identified and selected for comparative study. As shown inFIG. 3, these NIH3T3 and HCT-116 cell sets were plated in 96 well platesand evaluated for dose dependent cell killing by GCV for 4 days.Increasing GCV concentrations led to proportionally more cell killing inthe wild-type or Asn-125TK clones tested, and apparently both enzymesfunction similarly in regards to intracellular GCV metabolism andeffects on cell viabilities. For the Glu-125TK mutants, minimal cellkilling was observed in the NIH3T3 cells. However, the Glu-125TKexpressed in the HCT-116 cells led to minor decreases in cell viabilityat lower GCV concentrations, but at the highest concentration, 5 μM,cell viabilities dropped precipitously. For the Asp-125TK expressingHCT-116 cell lines, GCV had little effect on cell viability, althoughexpression of HSV-1 TKprotein was detected by antibody (data not shown).As expected, GCV had little effect on the cell viabilities of thenon-HSV-1 TK expressing parental cells.

EXAMPLE 18

Metabolic Labeling with [³H]GCV, [³H]Thymidine and [³H]dC

NIH3T3 fibroblasts and the human tumor cell line HCT-116 weretransfected with wild-type HSV-1TK or with one of three site specificmutants of amino acid Gln-125. From a panel of multiple HSV-1TKexpressing clones, a subset of clones from each cell line expressingwild-type HSV-1 TK, the Asn-125 HSV-1 TK mutant (Asn-125TK) and theGlu-125 HSV-1 TK mutant (Glu-125TK) were selected for comparativelyequivalent levels of HSV-1 TK protein expression based on Western blotdeterminations. In this study, these two sets of HSV-1 TK-expressing NIH3T3 and HCT-116 cell lines were evaluated for intracellular metabolismof [³H]GCV, [³H]thymidine, or [³H]dC. Cells were labeled with [³H]GCVfor 18 hours, then nucleotides were extracted in ice-cold 70% methanolas previously described (21, 24). The data in Table 3 summarize theamount of total nucleotide metabolites isolated in the methanol-solubleextracts (pmol/10⁶ cells) as well as the total amount ofmethanol-insoluble metabolites representative of incorporation into DNA.The Asn-125TK metabolizes GCV at (or near) equal levels to the wild-typeHSV-TK in both cell lines. The methanol-insoluble data, while only acrude indicator of [³H]GCV incorporation into DNA, reflects the numbersobtained with the soluble extracts. As compared with thenon-HSV-TK-expressing HCT-116 cells and consistent with data in theprevious study (24), minimal [³H]GCV metabolism was detected in theAsp-125TK cells and these cells were not further evaluated.

TABLE 3 Total Methanol Soluble and Insoluble [³H]-Metabolites (pmol/10⁶cells) NIH3T3 NIH3T3 NIH3T3 HCT116 HCT116 HCT116 HCT116 Cell Line[³H]GCV^(a,b) [³H]GvDNA^(c) [³H]GCV^(b) [³H]GvDNA^(c) [³H]T^(b)[³H]dC^(b) parent 2.8 2.8 1.1 1.2 2.4 1.1 Gln-125TK 498 48 1216 141 52.04.5 Asn-125TK 585 42 1085 133 8.0 1.2 Glu-125TK 3.6 2.6 33 27 3.3 0.9Asp-125TK N.D. N.D. 1.0 1.6 N.D. N.D. ^(a)Each value is the mean ofthree independent data points; N.D. = not determined^(b)Methanol-soluble nucleotide metabolites ^(c)Methanol-insolublemetabolites

The methanol-soluble metabolites were further separated into theirconstituent phosphorylated GCV metabolites by thin layer chromatography(21). As presented in Table 4, the predominant metabolite in eachHSV-TK-expressing cell line was GCVTP. In both HCT-116 and NIH3T3 celllines, the Asn-125TK cells indicated slightly higher levels of GCVTP ascompared to wild-type HSV-1 TK cell lines. The Glu125-TK in HCT-116cells resulted in a 23-fold or greater decrease in GCVTP levels, whilelevels of GCVTP in the NIH3T3 cells was only weakly detected. Thisdifference in the levels of GCVTP in the two Glu-125TK expressing celllines could explain the lack of sensitivity to GCV killing observed forthe NIH3T3 Glu-125TK cell lines.

TABLE 4 Phosphorylated Metabolites of GCV and Thymidine from HCT-116 andNIH3T3 Cell Clones. PHOSPHORYLATED METABOLITES (pmol/10⁶ cells) 3T3 3T33T3 116 116 116 116 116 GCVMP GCVDP GCVTP GCVMP GCVDP GCVTP TMP TTPparent 0.2 0 0 0.4 0 0.4 0.1 0.2 Gln-125TK 12 22 232 96 101 415 1.2 9.5WT Asn-125TK 11 21 272 95 104 533 0.2 0.9 Glu-125TK 0 0 1.6 3.0 2.5 180.2 0.4

Because the enzymatic data indicated that the Asn-125 and Glu-125mutations had altered deoxypyrimidine substrate utilization, themetabolism of thymidine and dC in the HCT-116 cell set were examined.Cells were grown to confluency and labeled with either [³H]thymidine or[³H]dC for 1 or 2 hours respectively prior to methanol extraction. Iflabeling was done in sub-confluent, dividing cultures, it was found thatthe metabolite numbers reflected cell growth rates, and thereforecellular kinase activities rather than that of HSV-TK activity (data notshown). As presented in the last two columns of Table 3, the levels ofdeoxypyrimidine metabolites extracted from the mutant HSV-TK cells wereanalogous to those isolated from parental HCT-116 cells rather than thewild-type HSV-TK cells. As shown in Table 4, the levels of TMP and TTPseparated from the methanol-soluble fractions of the [³H]thymidinelabeled HCT-116 Asn-125 TK and Glu-125TK cells were similar to parentalHCT116 cells rather than the wild-type HSV-1 TK expressing 116 cellline. These metabolite levels support the enzymatic data and highlightthe altered substrate specificities of the Glu-125 and Asn-125 HSV-1TKs. When expressed in cell lines, these mutant forms of HSV-1 TK appearto function more as GCV kinases rather than thymidine kinases.

EXAMPLE 19

Comparative GCV Sensitivities and Bystander Effect

Studies using an MTT cell viability assay determined that HCT-116 cellsexpressing the poor GCV metabolizing Glu-125 HSV-1 TK were just assensitive to GCV killing as the high-GCV metabolizing wild-type orAsn-125TK enzyme. Another striking aspect of this cell killing was thelarge number of apparently cytostatic, non-viable GCV-treatedGlu-125TK-expresssing cells that remained on the plate and did not stainwith MTT. Because the Glu-125TK expressed in NIH3T3 cells had littleeffect on their GCV sensitivities, only the HCT-116 cell panel wasevaluated in the rest of the study. Using the same panel ofHSV-TK-expressing HCT-116 cell lines normalized to one another andcharacterized for equivalent expression of HSV-1 TK protein, moresensitive clonal dilution assays were done. Cells previously plated in24 well plates were treated with GCV (0-10 μM) in triplicate for 24hours. Following drug removal, cells were diluted and replated in freshmedia from dilutions of 1:10 to 1:10,000. Surviving cell colonies werecounted 6-7 days later. As shown i n FIG. 4, 0.1 μM GCV treatment led toa 2-log decrease in the wild-type and Asn-125TK expressing cell colonynumbers. In the same cell lines treated with 10 μM GCV, a 4-log decreasein colony numbers were determined. In contrast, the Glu-125TK-expressingcells treated with 0.1 GCV μM caused no reduction in cell colonynumbers, while 1 μM or 10 μM GCV led to 0.7-log and 2.5-log decreases incolony numbers respectively. At higher concentrations of GCV (20-110μM), the the expressed Glu-125TK led to over 3-log reductions in colonynumbers (data not shown).

It has been previously established that HCT-116 cells expressing HSV-1TK are sensitive to bystander effect cell death via a connexin-43 gapjunction mediated transfer of GCV metabolites (24). To evaluate thiseffect in the HSV-1 TK 116 cell panel, clonal dilution assays wereperformed with different proportions of HSV-1 TK expressing cells(5-50%) mixed with HCT-116 parental cells. Cell populations were treatedwith 25 μM GCV for 24 hours, and then diluted from 1:10 to 1:10,000. Asshown in FIG. 5, only 5% wild-type or Asn-125TK-expressing cells wererequired to cause a greater than 1-log decrease in cell colony numbers.In these same cell lines, a greater than 4-log reduction in cell colonynumbers was detected with 25% and 50% proportions of HSV-1 TK-expressingcells. For the Glu-125TK-expressing cells, a 1-log GCV-mediatedbystander effect was observed at 10% proportions, and a near 3-logdecrease was detected with the 50% proportions. Even though thebystander effect with the Glu-125TK-expressing cells was clearlyattenuated relative to the other two cell lines, the Glu-125 mutant wasstill able to generate significant bystander effect cell killing.

EXAMPLE 20

Cell Cycle Analysis

GCV has also been previously reported to induce S and G₂-M phase cellcycle arrest in HSV-1 TK expressing glioma and melanoma cell lines(36-38). Therefore, the effect of 24 hr GCV treatment on the cellcycling of parental and the three HSV-1 TK-expressing cell lines wasexamined by flow cytometry of propidium iodide stained cells. As shownin FIGS. 6A and 6B and Table 5, GCV treatment of HCT-116 parental,non-HSV-1 TK-expressing cells had little effect on the percentage ofcells in each phase of the cell cycle as compared with untreated cells.In the wild-type and Asn-125TK-expressing cell lines, GCV treatment(FIGS. 6D and 6F) led to an increase in the proportion of cells in theS-phase, and undetectable percentages in G₂-M phase when compared tountreated cultures (FIGS. 6C and 6E). For the Glu-125TK-expressingcells, over 50% of the GCV treated cells were in S-phase and less than3% were in G₂-M (FIG. 6H). These results may reflect the more cytostaticeffects of GCV observed with the Glu-125TK-expressing cells.

TABLE 5 Cell Cycle Profiles of GCV Treated Cell Lines Cell Cycle Phase(%) Cell Line G₀/G₁ S G₂/M HCT-116 (−GCV) 43.3 35.0 21.7 HCT-116 (+GCV)39.0 44.0 17.1 WTGln-125TK (−GCV) 65.8 18.4 15.8 WTGln-125TK (+GCV) 60.139.9 0 Asn-125TK (−GCV) 63.4 18.0 18.6 Asn-125TK (+GCV) 70.0 30.0 0Glu-125TK (−GCV) 62.4  8.5 29.1 Glu-125TK (+GCV) 39.0 61.0 0

EXAMPLE 21

DAPI-Staining and Caspase 3 Apoptosis Assays

The differential dose responses, morphological features and cell cyclepatterns associated with the Glu-125 HSV-1 TK-expressing cells treatedwith GCV suggested induction of a distinct cell death mechanismdifferent from that observed in wild-type HSV-1 TK-expressing cells. Ithas been previously established that GCV treatment of wild-type HSV-1TK-expressing cell lines results in induction of apoptosis (36-37,39-40). Therefore, two late-stage apoptosis assays, nuclearDAPI-staining and caspase-3 activation, were done for GCV treatments ofthe three HSV-1 TK-expressing HCT-116 cell lines. As shown in FIG. 7,DAPI-stained nuclei of wild-type and Asn-125TK-expressing cells treatedwith GCV for 36 or 84 hrs indicated progressive increases in condensedand fragmented nuclei characteristic of apoptosis. Also, theDAPI-staining of these cell lines indicates a GCV-specific nuclearswelling of pre-apoptotic cells and enhanced staining of nucleoli. Thisnuclear swelling in response to GCV has been observed within 12 hours ofGCV administration in wild-type HSV-1TK HCT-116 cells (data not shown).For the Glu-125 HSV-1TK expressing cells, 36 hrs of GCV treatment led tofewer swelled nuclei and little evidence of apoptotic nuclei, althoughdistinct staining of condensed nucleoli was observed. Even after 84 hrsof GCV treatment of these cells, there were still comparatively fewerchanges in nuclear morphologies of the Glu-125TK cells compared to thewild-type or Asn-125 HSV-1TK expressing cells. There was apparentnuclear swelling in the Glu-125TK cells, and this reflects themorphological appearance of the cells observed in the MTT assays. Underidentical treatment conditions, GCV treatments of parental, non-HSV-1TKexpressing HCT-116 cells indicated none of the nuclear swelling orapoptotic fragmentations seen in the three HSV-1 TK-expressing celllines (data not shown).

A more direct analysis of apoptotic activity was done by assaying theactivation of the executioner protease, caspase 3. Activation of thezymogen form of caspase 3 has been implicated a s component of thelatter execution phase of apoptosis, and the substrate proteins cleavedby activated caspase 3 and related enzymes are responsible for theend-stage morphological and intracellular changes associated withapoptotic cell death (41-42). Caspase 3 activity was determined indifferent cell extracts derived from GCV-treated and control cells usinga calorimetric assay with the peptide substrate DVED (SEQ ID No. 4). Asshown in FIG. 8, the DVED-ase activity of GCV-treated Glu-125 HSV-1TKcells was three times lower than that observed for GCV-treated wild-typeor Asn-125 HSV-1TK-expressing cells. Co-incubation of GCV-treatedwild-type HSV-1 TK-expressing cells with the competing peptide DVED (SEQID No. 4) resulted in caspase 3 activities near untreated control cellvalues (data not shown). Thus, the results of the DAPI-staining andcaspase 3 assays are consistent with an altered apoptotic response andcell death pathway in GCV-treated Glu-125 HSV-1TK-expressing cells. Thecumulative results of this study are consistent with two distinct celldeath pathways induced by GCV treatment in the same HCT-116 cell linebackground that is dependent on the distinct enzymatic properties ofHSV-1 TK.

Discussion

Multiple X-ray crystal structures of HSV-1 TK have highlighted theimportance of Gln-125 in forming hydrogen bonds with pyrimidine andpurine substrates like GCV (13-17). It was demonstrated that fairlyconservative mutations of Gln-125 to Glu, Asp or Asn can have profoundeffects on substrate specficity and overall enzyme activity.Cumulatively, the data indicate that all three mutations appeardeficient in binding of TMP, which is the second substrate for the TMPkinase activity of wild-type HSV-TK. The inability of each mutant enzymeto photoincorporate the active-site directed photoaffinity analog,[³²P]5-azido-dUMP, further demonstrates this lack of TMP binding. It waspreviously determined that 5-azido-dUMP covalently crosslinks to anamino acid in the peptide comprising residues 112-132 (10). The lack ofphotolabeling of the mutant enzymes suggest that the site ofcrosslinking is at or near the vicinity of Gln-125. The Asn-125 mutantutilizes thymidine as a substrate poorly, but retains ganciclovir andacyclovir phosphorylation activities. Molecular modeling of the threemutations using a Flexidock program has predicted that loss of hydrogenbonding between thymidine and the Asp-125 or Asn-125 mutants contributesto the altered activities, while hydrogen bonding with each mutant andGCV is still retained. Clearly, the introduction of a negative charge(Glu, Asp) in the active site versus the more conserved Asn residue isanother contributing factor to the altered activities. When expressed inHCT-116 cell lines, the Asn-125 and Gln-125 HSV-1 TKs are as effectiveas wild-type HSV-1 TK at inducing GCV mediated cell killing.

A previous study evaluated the effect that single amino acidsubstitutions at Gln-125 (Asn, Asp, Glu, or Leu) had on thymidine andACV phosphorylation (17). In this study, it was reported that theirGlu-125 mutant had no detectable activities, and the Asn-125 mutant hada 50-fold and 3-fold increase in the K_(m)s of thymidine and ACVrespectively (17). These mutant HSV-1 TKs were not expressed inmammalian cell lines. These results differ significantly from the datapresented herein for the HSV-1 TK mutants. Other than the expression oftheir HSV-1 TK mutant enzymes as glutathione-S-transferase fusionconstructs (17), the reasons for the discrepancies in the results forthe same mutants is not clear. In other previous reports, a series ofrandom insertional oligonucleotide mutagenesis studies on HSV-1 TK havedemonstrated the catalytic role of the amino acids spanning residues159-172 (27-29), and many mutants have been identified which havealtered or improved substrate specificities for GCV and ACV (28, 29). Asa goal toward improving HSV-1 TK gene therapy strategies, some of themutant HSV-1 TKs generated by random insertional mutagenesis were testedfor cell killing efficacy in mammalian cells (29). One of these mutants,which had four changed residues (Ile-160 to Leu; Phe-161 to Leu; Ala-168to Val; Leu-169 to Met), was shown to have 43-fold and 20-fold greatersensitivities to cell killing with GCV and ACV respectively (29). TheK_(m) of GCV for this mutant was 5-fold lower than wild-type HSV-1 TK,and the kcat of the mutant enzyme for thymidine, ACV and GCV remainedthe same as wild-type HSV-1 TK (29). Because these amino acid changesoccur in a distinct catalytic region of HSV-1 TK and the Gln-125 appearsto be only involved in nucleoside base binding (13-17), it should bepossible to construct hybrid HSV-1 TKs comprising mutations from bothsites to generate an enzyme with minimal TK/TMPK activities and maximalacyclic purine nucleoside phosphorylation activities.

In other cell culture studies, it has been demonstrated that the moreHSV-1 TK protein expressed in a cell, the more efficient GCV metabolismand cell killing are (30-33). Whether by improving expression orcatalytic efficiencies, these cumulative results for HSV-1 TK indicatethat any method of increasing GCV metabolism could result in increasedtherapeutic benefits. Because the K_(m) for thymidine is over 70-100times lower than that of GCV for wild-type HSV-TK (29), it washypothesized that in a cellular environment the Asn-125 mutant would actprimarily as a GCV kinase, particularly as thymidine and its metaboliteswill compete less with GCV for binding in the active-site. This appearsto be the case in both cell lines tested for the Asn-125 mutant, andeven in the HCT-116 cells expressing the Glu-125 mutant. The GCVmetabolism properties and mechanistic aspects of GCV cell killing by theAsn-125 and Glu-125 mutants in the HCT-116 cells are further evaluated.These types of HSV-1 TK mutants described herein will allow theevaluation of whether the TK/TKMP activities of HSV-1 TK cause anycellular problems related to altered nucleotide metabolism and poolsizes. This could be especially important in the cancer gene therapytrials for myeloma (34) and leukemia (35) that administer HSV-1TK-expressing T-lymphocytes to patients for immune protection andsurveillance following bone marrow transplants. HSV-1 TK acts as asafety gene in these studies to allow termination of the treatment viaGCV if graph-versus-host disease develops, thus use of an HSV-1 TK thatis predominantly a GCV kinase could prove to be safer and moreeffective. Efforts to characterize the expression and metabolism of GCVin T-lymphocytes expressing Glu-125 and Asn-125 HSV-1 TKs are currentlyin progress.

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Any patents or publications mentioned in this specification areindicative of the levels of those skilled in the art to which theinvention pertains. Further, these patents and publications areincorporated by reference herein to the same extent as if eachindividual publication was specifically and individually indicated to beincorporated by reference.

One skilled in the art will appreciate readily that the presentinvention is well adapted to carry out the objects and obtain the endsand advantages mentioned, as well as those objects, ends and advantagesinherent herein. The present examples, along with the methods,procedures, treatments, molecules, and specific compounds describedherein are presently representative of preferred embodiments, areexemplary, and are not intended as limitations on the scope of theinvention. Changes therein and other uses will occur to those skilled inthe art which are encompassed within the spirit of the invention asdefined by the scope of the claims.

                   #             SEQUENCE LISTING<160> NUMBER OF SEQ ID NOS: 4 <210> SEQ ID NO 1 <211> LENGTH: 21<212> TYPE: DNA <213> ORGANISM: artificial sequence <220> FEATURE:<221> NAME/KEY: primer_bind<223> OTHER INFORMATION: a mutagenic oligonucleotide  #primer used to      replace Gln-125 with Asp-125 <400> SEQUENCE: 1acaagcgccg acataacaat g            #                  #                   #21 <210> SEQ ID NO 2 <211> LENGTH: 21<212> TYPE: DNA <213> ORGANISM: artificial sequence <220> FEATURE:<221> NAME/KEY: primer_bind<223> OTHER INFORMATION: a mutagenic oligonucleotide  #primer used to      replace Gln-125 with Asn-125 <400> SEQUENCE: 2acaagcgcca acataacaat g            #                  #                   #21 <210> SEQ ID NO 3 <211> LENGTH: 21<212> TYPE: DNA <213> ORGANISM: artificial sequence <220> FEATURE:<221> NAME/KEY: primer_bind<223> OTHER INFORMATION: a mutagenic oligonucleotide  #primer used to      replace Gln-125 with Glu-125 <400> SEQUENCE: 3acaagcgccg aaataacaat g            #                  #                   #21 <210> SEQ ID NO 4 <211> LENGTH: 4<212> TYPE: PRT <213> ORGANISM: artificial sequence <220> FEATURE:<223> OTHER INFORMATION: a peptide substrate used  #for a colorimetric      assay <400> SEQUENCE: 4 Asp Val Glu Asp

What is claimed is:
 1. A method of killing target cells in an individualin need of such treatment, comprising the steps of: (a) transfecting ortransducing said target cells with a gene encoding a mutant herpessimplex virus type 1 thymidine kinase protein, wherein said proteincontains a site-specific mutation at amino acid position 125 of wildtype herpes simplex virus type 1 thymidine kinase protein, wherein saidamino acid at position 125 is glutamine, and wherein said site-specificmutation is from glutamine to asparagine or glutamic acid, wherein saidgene is directly injected into said target cells; and (b) contactingsaid transfected or transduced target cells with an effective amount ofa prodrug, wherein said prodrug is a substrate for the mutant herpessimplex virus type 1 thymidine kinase to produce a toxic substance,wherein said toxic substance kills said transfected or transduced targetcells.
 2. The method of claim 1, wherein said prodrug is administeredsystemically or locally.
 3. The method of claim 1, wherein said prodrugis selected from the group consisting of nucleoside analog acyclovir,ganciclovir and 5-bromovinyldeoxyuridine.
 4. The method of claim 1,wherein said target cells are selected from the group consisting oftumor cells and virally infected cells.